The subject matter is directed to systems and methods for determining the size of a molecule, or, more generally, the distribution of sizes of an ensemble of molecules.
Gel electrophoresis is the most commonly used technique for measuring distributions of molecular sizes. In its usual application to proteins, the proteins are denatured so the electrophoretic mobility measures the molecular length, and thereby the approximate molecular weight. Native gel electrophoresis can also be applied to biomolecules in their functional conformation, though the interpretation of mobilities in native gels is often ambiguous. Gel electrophoresis cannot be applied to large or weakly associating molecular complexes. Additionally, gel electrophoresis typically requires several hours to run, requires large amounts of sample, and consumes ˜100 mL of reagents.
The sizes of particles larger than ˜10 nm diameter can be determined by dynamic light scattering (DLS). All particles scatter light, so dust or impurities confound or interfere with DLS measurements, and DLS can only be applied to the major component in a heterogeneous mixture. Furthermore, the inverse Laplace transform used in interpretation of DLS is subject to noise, so DLS measurements are often imprecise.
Fluorescence correlation spectroscopy (FCS) measures the size of fluorescent species in solution by measuring the distribution of residence times in a focused laser spot. FCS works best for small molecules, with hydrodynamic radii of less than 20 nm. As with DLS, FCS cannot easily distinguish between individual particles and fluorescent aggregates. FCS provides only a very coarse measure of molecular size, and is not well suited to measuring heterogeneous size distributions.